Radiation leads to cells arresting or delaying in the G2 phase of the cell cycle. Here we propose to examine the effects of radiation on a late event of the normal G2 phase, that of centrosome separation. As cells enter mitosis, a spindle is formed contributing to chromosome alignment and segregration. The centrosomes form the end of the bipolar spindle and are required for accurate chromosome segregation and for cell division. The centrosome is duplicated during S phase and remains paired as cells enter G2. As cells exit G2 the centrosomes separate and move to opposite poles as part of the mitotic spindle. We propose in this application to explore the mechanisms through which radiation affects the centrosomes. We will examine both the effect of radiation on the centrosome during the G2 block and subsequently the resumption of the cell cycle. We will examine the effects of radiation on nek2, a kinase, and the phosphatases, protein phosphatase 1 and cdc14 all known to be involved in the separation of the centrosome. After completion of G2, irradiated cells often resume the cell cycle, but depending on the dose of radiation may ultimately fail to survive. An examination of centrosomal functions in irradiated cells exiting from the division block has revealed that many cells fail both in organization of centrosomes as well as failure of cytokinesis. This may account for the increased frequency of multinucleated cells, chromosomal missegregation and even cell death seen after irradiation. Thus it is our intention to examine centrosome function in irradiated cells, both immediately during the division delay and later as cells reinitiate cell division. We will ask whether molecules implicated in completion of mitosis are altered in activity, amount or localization by irradiation and ask how this affects the ability of the cells to resume correct cycling.